Novel muscle-derived extracellular matrix hydrogel promotes angiogenesis and neurogenesis in volumetric muscle loss

Abstract

Volumetric muscle loss (VML) represents a clinical challenge due to the limited regenerative capacity of skeletal muscle. Most often, it results in scar tissue formation and loss of function, which cannot be prevented by current therapies. Decellularized extracellular matrix (DEM) has emerged as a native biomaterial for the enhancement of tissue repair. Here, we report the generation and characterization of hydrogels derived from DEM prepared from WT or thrombospondin (TSP)-2 null muscle tissue. TSP2-null hydrogels, when compared to WT, displayed altered architecture, protein composition, and biomechanical propertied and allowed enhanced invasion of C2C12 myocytes and chord formation by endothelial cells. They also displayed enhanced cell invasion, innervation, and angiogenesis following subcutaneous implantation. To evaluate their regenerative capacity, WT or TSP-2 null hydrogels were used to treat VML injury to tibialis anterior muscles and the latter induced greater recruitment of repair cells, innervation, and blood vessel formation and reduced inflammation. Taken together, these observations indicate that TSP2-null hydrogels enhance angiogenesis and promote muscle repair in a VML model.

Introduction

Irreversible volumetric muscle loss (VML) affects the lives of healthy as well as diseased individuals[1]. For example, accidental blunt musculoskeletal injuries from military casualties and thermal and radiation treatment in cancer patients both result in the loss of muscle structure and function, leading to necrosis[2–4]. In addition, VML, caused by congenital malformations, traumatic injuries, surgical ablations, or degenerative myopathies, remains a challenge in the medical setting despite the regenerative capacity of muscles[5, 6]. Thus, there is a critical need to develop effective strategies toward regeneration of muscle and functional restoration[1]. Extracellular matrix (ECM) is an essential component of the stem cell niche through its modulation of multiple stem cell functions[7, 8]. In VML, extensive loss of ECM prevents complete regeneration and leads to irreversible damage and chronic functional deficits[9].

Injured muscles have the capacity to regenerate due to action of satellite stem cells that can both self-renew and give rise to committed myogenic progenitors[10]. Importantly, the regenerative capacity of resident satellite cells is dependent on their well-characterized “niche,” located between the sarcolemma of individual myofibers[5, 8, 10]. Therefore, ECM and ECM-like constructs are considered as suitable treatments to enhance repair. In fact, a number of native or synthetic constructs have been prepared following the removal of cellular components and have been approved by the Food and Drug Administration for clinical use [11, 12].

Skeletal muscle is the most abundant tissue in the body and is composed of muscle fibers, muscle stem cells, nerves, blood vessels, interstitial cells and unique ECM containing various components, such as glycosaminoglycans (GAGs), proteoglycans, collagens and growth factors[1]. Therefore, muscle can be considered a source for ECM to treat muscle injuries. Previously, skeletal muscle ECM from mongrel dogs’ processed by enzymatic and chemical decellularization was shown to contain growth factors, glycosaminoglycans, and basement membrane structural proteins[13, 14]. Other studies have described the decellularization and preparation of muscle-derived hydrogels from porcine, human, rat, bovine and rabbit sources with various degrees of ECM preservation[15–19]. Finally, muscles were isolated and processed from four different pigs to evaluate animal to animal variability and to investigate fabrication approaches for ECM hydrogels[20]. Specifically, harvesting conditions were shown to influence hydrogel composition with varying amounts of fat and proteins. In addition, a dose optimization study in rats, showed that there is an optimal ECM concentration to induce recovery in an aged mouse hindlimb ischemia model[21].

ECM composition and structure vary based on tissue origin and play important roles in guiding responses to injury and surgical materials based on ECM are used in the clinic[22, 23]. For example, ECMs derived from multiple species and organs have been applied for repair of tendon and oral cavity as well as soft tissue augmentation[23]. In addition, ECMs derived from small intestinal submucosa have been widely used to repair abdominal and thoracic wall, muscle flap defects, and soft tissue reinforcement and myocardial matrix-derived hydrogels to aid in left ventricular (LV) remodeling in chronic myocardial infarction (MI)[24].

Native hydrogels are advantageous in that they retain ECM properties but are difficult to engineer and customize with precision[1, 25]. Specifically, many studies utilized pepsin digestion coupled with varying ECM concentrations and crosslink temperatures to produce hydrogels[15, 16, 26]. Other studies focused on the selective removal of ECM components or the addition of exogenous factors[27, 28]. In a different approach, investigators modified ECM hydrogels by including methacrylate to generate microporous structure and enhance early cellularization and endothelialization [29]. In addition, manipulation of source cells and tissues to exert more control over hydrogel properties has been pursued[30, 31]. For example, the overexpression of growth factors by cells secreting ECM on a hydroxyapatite scaffold resulted in a matrix coating enriched in these factors, demonstrating the feasibility of a bottom-up approach[31]. We also showed that hydrogels derived from the skin of mice lacking the matricellular protein thrombospondin-2 (TSP-2), had altered ECM composition, assembly, and biomechanical properties and could enhance healing of wounds[32]. Thus, we were able to induce favorable responses including angiogenesis without the need for additional modifications of the native hydrogel. These observations were consistent with the known anti-angiogenic property of TSP2 and its role in ECM formation and expression post-injury in remodeling tissues[33, 34].

In the present study, we undertook a decellularization approach based on enzymatic digestion to prepare hydrogels from the skeletal muscle of WT and TSP2 KO mice. We also conducted a thorough characterization on gel properties including scanning electron microscopy (SEM), proteomics, and rheometric analysis, to identify difference between WT and TSP2KO ECM. In vitro and in vivo studies revealed enhanced cell-ECM interactions and enhanced cell migration in TSP2-KO derived hydrogels. Finally, in a VML model, WT mice treated with TSP2 KO hydrogel showed enhanced repair when compared to those treated with WT hydrogel. These findings demonstrate the healing capacity of TSP-2 KO hydrogel following a single application and suggest its translational potential.

Results

Preparation and characterization of muscle ECM hydrogels.

WT and TSP2 KO muscle ECM hydrogels were prepared via a stepwise process outlined in Fig. 1A with a focus on preventing ECM damage and preserving proteins. To confirm efficient decellularization and maintenance of ECM architecture, processed tissues were stained with H&E and Masson’s trichrome (Fig. 1B), analyzed for DNA content (Fig. 1C), and protein content (Supplementary Fig. 1A–C). Histological analysis revealed the lack of cells, and this was supported by the low DNA content in decellularized ECM and hydrogels. Specifically, WT and TSP2 KO hydrogels contained (2.14 ± 0.99 ng/mg) and (2.54 ± 1.41 ng/mg), respectively, which is far below the suggested threshold of 50 ng DNA/mg ECM for decellularized constructs [34]. Moreover, SDS–poly-acrylamide gel electrophoresis (SDS–PAGE) revealed the retention of proteins during the decellularization process that was similar between WT and TSP2 KO samples (Supplemental Fig. 1A). Western blot analyses of hydrogel protein extracts with anti-TSP2 or anti-Collagen antibodies confirmed the absence of TSP2 in TSP2KO hydrogels (Supplemental Fig. 1B) and the presence of the α (115~130 kD), β (~200 kD), γ (>250 kD) chains of collagen in both samples (Supplemental Fig. 1C). Additionally, proteomic analysis identified 10 extracellular matrix proteins that were differentially expressed between WT and TSP2KO muscle ECM (Fig. 1H–J). The majority of these were collagen-related proteins (Fig. 1H). Specifically, Col2a1 and Col11a2 were highly expressed in the KO samples while Col3a1, Col4a4, Col5a1, and Col5a2 were lower (*p<0.05, Fig. 1H, J). However, the major structural proteins like Collagen type I showed similar expressional level between the two genotypes (Fig. 1H–J).

Figure 1. Fabrication and characterization of muscle ECM hydrogels.

(A) Procedure of preparing muscle ECM hydrogel. Muscle ECM was lyophilized, and the resulting hydrogel concentration was 10 mg ECM/mL. (B) Tissue samples were stained at various decellularization steps by H&E and Trichrome staining, to depict the removal of muscle cells throughout the process and the retention of collagen proteins. (C) DNA content was tested by the DNeasy Kit and NanoDrop and a significant difference was detected after decellularization for both WT and TSP2KO samples (***p < 0.001, n=5). (D) Microstructure observation of the muscle hydrogels by SEM. Analysis of the fibril diameter in the WT and TSP2KO hydrogels indicated that they were similar. (F) Analysis of the fibril curvature in the WT and TSP2KO hydrogels indicated the average curvature of TSP2KO fibrils was higher than that of WT fibrils (*p < 0.05, n = 6). (G) The storage modulus for both hydrogels at 10mg/mL indicated that the WT hydrogel was more elastic than the TSP2KO hydrogel (*p < 0.05, n = 6). (H) Collagen abundance from quantitative proteomics was found to differ between WT and TSP2KO mouse muscle ECM. (I) A proteomic volcano plot demonstrated differences between genotypes (only ECM proteins showed difference are denoted in the plot). (J) The top 26 most abundant ECM proteins showed differences between WT and TSP2 KO muscle hydrogels. Results are given as mean + SEM, n = 3, *p < 0.05.

Prepared hydrogels were also visualized by SEM as shown in Fig. 1D. Image analysis revealed that TSP2 KO hydrogels contained collagen fibrils that were equal to WT in diameter but displayed greater curvature (Fig. 1 E–F). Finally, rheology showed that TSP2 KO hydrogels had altered biomechanical properties with a lower storage modulus (Fig. 1G, Supplementary Fig. 1D).

Cell interactions with muscle ECM hydrogels.

To assess the ability of hydrogels to support cell functions relevant to responses to injury, we analyzed the morphology, proliferation and migration of C2C12 on WT or TSP2 KO hydrogels (10 mg/ml). No differences in proliferation were detected (Supplemental Fig. 2A–B). SEM visualization and image analysis revealed that C2C12 cells spread faster on TSP2 KO hydrogels and by 4 h displayed larger cell area and perimeter and were less circular and more elongated in shape (Fig. 2 A–B). Cell migration was investigated in a modified transwell assay where C2C12 were placed on pre-formed hydrogels and their invasion into the hydrogels was measured up to 72 h by histological analysis as shown in Fig. 2C. Measurements revealed greater invasion of C2C12 cells into TSP2 KO hydrogels after 72 h (Fig. 2 D).

Figure 2. Cell-hydrogel interactions.

(A) Representative SEM images of C2C12 cells seeded on WT or TSP2 KO hydrogels for 2 and 4 h. (B) Cells on TSP2 KO hydrogels for 4 h exhibited larger area and perimeter and were more elongated. One-way ANOVA with post hoc Turkey HSD test, n = 50 cells, ***p < 0.001. (C) Representative images of H&E-stained sections following seeding of cells on hydrogels in a transwell system for 72 h. (D) Cell invasion was measured at the indicated time points and found to be greater in TSP2 KO hydrogels at 72 h (*p < 0.05, n = 3). (E) Representative merged fluorescent images of HUVECs cultured on hydrogels for 24 h and stained with DAPI (blue; nuclei), and rhodamine-phalloidin (red; actin cytoskeleton). (F, G) Image analysis indicated that tube formation was greater on TSP2KO hydrogels (*p < 0.05, n = 4).

To investigate the responses of other cell types, we utilized NIH3T3 cells as a suitable fibroblast cell type. Similar to C2C12 cells, we did not observed differences in proliferation when cultured on WT or TSP2 KO hydrogels (Supplemental Fig. 2C–D). To evaluate migration, NIH3T3 were evaluated as described above with the exception that the concentration of hydrogel was lowered to 2mg/ml due to insufficient invasion at higher hydrogel densities (Supplementary Fig. 2F). Measurements revealed increased migration into TSP2 KO hydrogels at 12 h post plating (Supplemental Fig. 2 F–G). SEM analysis at 2 h post plating showed that NIH3T3 cells were more spread and displayed extensive interactions and integration with the ECM (Supplementary Fig. 2H).

To further explore cell-hydrogel interactions relevant to injury responses, we utilized human umbilical vein endothelial cells (HUVEC) and macrophages (murine immortalized bone marrow-derived macrophages, iBMM) to probe possible angiogenic and immune responses, respectively. Proliferation and morphology of HUVEC was monitored for 72 h and was found to be similar on both hydrogels (Supplemental Fig. 3 A–B). However, when mixed within WT or TSP2 KO hydrogels in a tube formation assay, HUVEC formed a greater number of tubes of similar diameter in the latter (Fig. 2 E–F). iBMM cultured on either WT OR TSP2 KO hydrogels for up to 14 days exhibited similar densities and morphology (Supplementary Fig. 3C). Possible activation and polarization of macrophages during the culture period was monitored by analyzing the expression of key cytokines and growth factors (CD 86, IL-1 β, TNF-α, TGF-β, IL-10, and Arg1) by quantitative Reverse Transcription - Polymerase Chain Reaction (qRT-PCR) and was found to be similar (Supplementary Fig. 3D). These in vitro observations suggested that when compared to WT, TSP2 KO hydrogels could increase angiogenesis without altering inflammatory responses.

Foreign body response of muscle ECM hydrogels.

Subcutaneous (SC) injections were performed to assess the overall foreign body response of WT and TSP2 KO hydrogels. Specifically, ECM solutions were prepared and injected SC into mice to form hydrogels in situ, which were retrieved 5 or 10 d later. Histological evaluation following H&E stain of hydrogel sections revealed the progressive decrease in observable hydrogel area with the TSP2 KO hydrogels displaying more elongated shape (Fig. 3 A–B). In addition, more cells were observed within TSP2 KO hydrogels at d 5 (Fig. 3 C and Supplemental Fig. 4 A). Immunohistochemistry was employed to identify vimentin-positive cells as well as macrophages (F4/80 Ab) and T-cells (pan T-cell CD3e Ab) (Fig. 3D). Histomorphometric f analyses revealed that TSP2 KO hydrogels contained higher number of vimentin-positive cells (d 5 and d 10) and T-cells (d 5) (Fig. 3E). No differences in the number of macrophages were observed, which is consistent with our in vitro observations.

Figure 3. Evaluation of WT and TSP2KO muscle-derived hydrogels foreign body response in subcutaneous injection model.

(A) Representative images of H&E-stained sections of WT and TSP2 KO hydrogels injected SC in WT mice for 5 or 10 days. (B) Semi-quantitative image analysis of hydrogel area revealed that the TSP2KO hydrogels had a more elongated shape (**p < 0.001, n = 5). (C) Image analysis revealed that TSP2KO hydrogel contained more cells at day 5 in comparison to WT (*p < 0.05, n = 5). (D) Representative images of day 5 and day 10 hydrogel sections stained with Vimentin (Fibroblast/mesenchymal cells), F4/80 (macrophages), or CD3e (T cells) and visualized with the peroxidase reaction. (E) Image analysis revealed increased Vimentin + cells on day 5 and 10, no differences in F4/80 + cells, and increased CD3e+ cells on day 5 in TSP2 KO hydrogels. (F) Representative images of WT and TSP2 KO hydrogels injected SC in WT mice for 10 days and stained with antibodies to detect neurofilament H as a marker for innervation. In contrast to TSP2 KO, no innervation was detected in WT hydrogels. (G) Representative single and merged immunofluorescence images show the presence and absence of newly formed vessels in day 10 TSP2 KO and WT hydrogels, respectively. (**p < 0.01 at day 5, *p < 0.05 at day 10, n = 5).

Histological examination suggested an angiogenic response within TSP2 KO but not WT hydrogels and immunohistochemistry was employed to detect newly formed blood vessels. Specifically, d 10 sections were stained with antibodies to CD31 and α-SMA to detect endothelial cells and smooth muscle cells, respectively. Consistent with the histological assessment, newly formed CD31 and α-SMA-positive vessels were observed only in TSP2 KO hydrogels (Fig. 3G). To evaluate whether neovascularization of hydrogels was coupled with nerve formation, sections were also stained with Neurofilament H Ab. Consistent with the observation of an angiogenic response, newly formed nerves were only detected in TSP2 KO hydrogels (Fig. 3F). Taken together, these observations suggested that both WT and TSP2 KO hydrogels were well tolerated and did not induce excessive inflammation. However, the latter exhibited greater cell content with newly formed vessels and nerves indicative of regenerative capacity.

Application of hydrogel in VML model.

VML model was created to evaluate muscle hydrogel function following surgical injury. Specifically, WT or TSP2 KO preformed hydrogels were placed in a surgical defect and then the muscle and skin were sutured as shown in Supplemental Fig. 5 A. Tissue samples were obtained on day 3, 14 or 28 post injury and analyzed via Trichrome and Sirius red staining (Fig. 4A, Supplementary Fig. 5B). Observable hydrogel area (blue stain) was measured using Image J and was found to be decreased, via a semi-quantitative assay, at d 14 in the case of the TSP2 KO (Fig. 4 B). It should be noted that this stain cannot distinguish between the hydrogel and native and newly formed cell-derived ECM. Importantly, picrosirius red staining suggested TSP2KO hydrogel degradation did not elicit fibrosis. However, muscle healing in untreated VML mice was much slower with prominent fibrosis (Fig. S5). Samples were also stained with H&E, which showed that TSP2 KO hydrogels contained more cells at d 3. In addition, immunohistochemical detection of laminin revealed remodeling of the hydrogels with increased deposition at d 14. Immunohistochemical analysis revealed the enhanced presence of vimentin-positive cells (mesenchymal lineage, fibroblasts) in TSP2 KO hydrogels (Fig. 4A, C). TSP2 KO hydrogels also contained less macrophages at d 14, while no difference in the number of CD3e-positive (Pan T cells) was observed (Fig. 4A, D, E).

Figure 4. VML model and treatment with muscle-derived hydrogel.

(A) Representative images of WT and TSP2 KO hydrogels 3 and 14 days after application to injured muscles and stained with Masson’ trichrome, Sirius red, H&E, antibodies to detect Laminin, Vimentin, F4/80 or CD3e. (B) Semi-quantitative image analysis showed a reduction of the observable TSP2 KO hydrogel area at 14 d (*p < 0.05, n = 3). (C-E) The number of cells were analyzed via ImageJ. (C) There was no difference in the presence of fibroblast between the TSP2KO and WT gels for each time point (p > 0.05, n = 3). (D) There were fewer macrophages present in the TSP2KO gel as compared to the WT gel at day 14 (*p < 0.05, n = 3). (E) No T cells were present in either of the hydrogels 3 days after transplantation. At day 14, the total presence of T cells did not differ for either hydrogel (p > 0.05, n = 3). Stars (☆) indicate the hydrogel.

To further evaluate hydrogel-induced remodeling, samples were stained with CD31 and α-SMA Abs to analyze newly formed blood vessels and neurofilament H Ab to detect innervation. Consistent with enhanced presence of repair cells, we detected an increase in mature (α-SMA - positive) blood vessels and innervation in TSP2KO hydrogels at d 14 (Fig. 5 A–C). Additionally, detection of Laminin and satellite cells (PAX7) by immunofluorescence suggested ongoing remodeling at 14 d (Fig. 5D). No difference was observed between WT and TSP2KO hydrogel treated muscle in terms of PAX7 positive area (Fig. 5E). Increase in neurofilament H was confirmed by western blot analysis of muscle hydrogel extracts (Supplementary Fig. 5C). Taken together, these observations suggest that mice treated with TSP2KO hydrogel induce greater healing with enhanced neovascularization and innervation.

Figure 5. Detection of innervation and angiogenesis in the VML model.

(A) Representative images of injured muscles treated with WT or TSP2 KO hydrogels for 14 days and stained with antibodies to detect α-SMA, or CD31 as markers of vessel formation. (B) Quantification of vessel formation by image analysis revealed an increase in muscles treated with TSP2KO hydrogel at day 14 (**p < 0.01, n=5). (C) Representative images of muscles treated with WT or TSP2 KO hydrogels for 14 days and stained with antibodies to detect neurofilament H as a marker for innervation. In contrast to TSP2 KO, no innervation was detected in WT hydrogels (D) Representative images of muscle treated with WT or TSP2KO hydrogels for 14 days and stained with antibodies of Laminin and PAX7 to detect remodeling and skeletal muscle satellite cells, respectively (arrows indicate PAX7 positive cells). (E) Quantification of PAX7 positive areas (%) in the muscle tissues treated with WT or TSP2KO hydrogels (n=3).

Discussion

In the present study, we explore muscle-derived hydrogels from WT and TSP2KO mice and show that the latter can induce greater regeneration in a VML model. Severity of injury in VML overwhelms regenerative processes leading to limited repair and utilization of this model allowed rigorous evaluation of the hydrogel’s therapeutic potential[9]. Biomaterials with or without cells represent an attractive treatment and native hydrogels have several advantages including tunability and matching the composition of damaged tissue. It has been suggested that suitable biomaterials should be biocompatible, biodegradable and capable of encouraging cell growth and incorporation with surrounding tissue including formation of new vessels and peripheral nerves[35, 36]. Here, we show that hydrogels derived from TSP2 KO muscle fulfill these criteria in a VML model. Previously, the loss of TSP-2 was shown to be associated with the production of collagen fibrils and fibers that were irregular in shape and size, suggesting that it plays a role in collagen fibrillogenesis and ECM assembly[34]. Moreover, ECM derived from TSP-2 null skin was shown to have altered matrix structure and biomechanical properties and was able to enhance wound healing[32, 37]. Consistent with these previous studies, ECM hydrogel derived from TSP2 KO muscle was shown to share these alterations including having more curved collagen fibrils and lower elastic modulus than WT. In vitro studies with myocytes, NIH3T3, and endothelial cells demonstrated increased spreading, migration, invasion, and tube formation in TSP2 KO hydrogels. Interestingly, these changes were not due to effects on proliferation, suggesting the ability of TSP2 KO hydrogels to influence distinct cell functions. These effects were also cell-type specific because macrophages did not display distinct behaviors in terms of morphology or activation when cultured on WT or TSP2 KO hydrogels.

Both WT and TSP2 KO hydrogels were injected SC and the latter contained a greater number of cells, blood vessels, and neurofilament H-positive structures. Immunohistochemistry revealed an increase in vimentin-positive cells suggesting greater fibroblast invasion. Consistent with in vitro observations, there was no difference in the number of macrophages but TSP2 KO hydrogels did contain more T cells at d 5. T cells have been implicated in the VML model and the response to biomaterials[38]. Moreover, a recent study involving delayed Interleukin-10 (IL-10) delivery in the VML model implicated T_reg cells in promoting repair[39]. However, the number of T cells detected in our study by immunohistochemistry was very small (~5 T cells vs ~100 macrophages and ~1,000 vimentin-positive cells/mm²). Therefore, it is unclear if the increase in these cells contributes to the enhanced cell infiltration, angiogenesis, and innervation. Importantly, enhanced angiogenesis in TSP2 KO hydrogels is consistent with numerous observations in mice and mice treated with TSP2 KO-derived skin-derived ECM[32, 37]. Because TSP2 can also function as an inhibitor of angiogenesis, it is possible that the pro-angiogenic effect is both direct and indirect involving TSP2 interactions with CD36 and/or CD47 on endothelial cells, increased soluble vascular endothelial growth factor (VEGF), or altered endothelial cells (EC)-ECM interactions[34].

TSP2 KO hydrogels also displayed innervation evidenced by the presence of neurofilament H-positive structures. Formation of such structures was not observed in WT hydrogels, suggesting a correlation between neovascularization and innervation in TSP2 KO hydrogels. As mentioned above the loss of TSP2 has been extensively associated with neovessel formation in multiple injury models. However, the impact, if any, of TSP2 deficiency on hydrogel innervation is not known. It is also appreciated that in the context of tissue repair, angiogenesis is linked to nerve formation in a process known as neurovascular regeneration, which is thought to be critical for functional recovery in VML[35]. Unlike in tissues like skin[40], the association of these process and the nature of neurovascular communication in the regeneration of skeletal muscle are poorly understood. Nevertheless, it is appreciated that revascularization plays an important role in peripheral nerve regeneration and multiple factors, like VEGF, ephrins, and neuropilins, have been implicated in both processes[35, 41]. Importantly, these improvements are achieved with a single application of hydrogel following injury. Numerous studies have described treatments to improve outcomes in the VML model, reviewed in ([42, 43]). Unlike the approach described here, these studies involve the inclusion of cell and/or therapeutics agents such as stem cells, immune cells, and biological factors, which necessitates complex fabrication and administration.

In the present study, we developed a novel TSP2KO mice muscle-derived ECM hydrogel that demonstrated a more curved fibril structure and unique mechanical properties. This hydrogel was able to better support C2C12 and NIH3T3 cell adhesion and invasion, HUVEC in vitro tube formation, and in vivo microvascular and nerve formation. Furthermore, it was evident that mice with TSP2KO hydrogels recovered better from muscle damage following VML surgery. This genetically modified procedure offers a new mechanism for modifying native hydrogels. In addition to matrix hydrogels from TSP2 KO mice, we envision that this genetic approach could also be coupled with pharmacological or other approaches and expanded to larger animal models.

Materials and Methods

Isolation and Decellularization of Muscle.

All procedures involving animal use were approved by the Animal Care and Use Committee of Yale University and abided by the regulations adopted by the National Institutes of Health. 12–14-week-old C57BL/6J WT or TSP2 KO mice were euthanized by CO₂ inhalation. Skeletal leg muscles were excised and washed with with ddH₂O and then incubated for 6 h in 0.25% Trypsin-EDTA (Gibco), followed by three 15 min washes in ddH₂O and incubations in 70% ethanol for 12 h and 3% H₂O₂ (J.T. Baker) for 15 min and finally two 15 min washes in ddH₂O. They were then incubated overnight in 1% Triton X-100 (American Bioanalytical) in 0.26% Tris (American Bioanalytical)/0.69% EDTA (Sigma). They were incubated for 6 h in 1% Triton X-100 /0.2% Sodium deoxycholate (SDC, Sigma) and terminally sterilized in 0.1% peracetic acid (Sigma) in 4% ethanol for 2 h. Finally, they were subjected to six 15 min washes in sterilized ddH₂O each, as described previously [44, 45]. All the above steps were performed at room temperature on an orbital shaker. Afterward, decellularized tissues lyophilized and stored at −80°C until use. To monitor decellularization and tissue integrity, samples from each step were fixed with z-fix (Thermo Fisher Scientific) and stained with hematoxylin and eosin (H&E) and Trichrome staining.

Hydrogel Preparation.

ECM derived from WT or KO muscle was digested in a solution of 1 mg/mL pepsin (Sigma) in 0.01 N HCl (J.T. Baker) for 72 h (10 mg dry weight ECM per milliliter of pepsin solution). The solubilized ECM was neutralized and buffered with sodium hydroxide (1/10 digest volume) and 10× phosphate-buffered saline (PBS) (1/9 digest volume), respectively. To prepare ECM-derived hydrogels, buffered and solubilized ECM was diluted to a final concentration of 10 mg/mL with PBS to form the pre-gel solution and stored on ice until use.

Quantitative Detection of DNA and Protein Content.

The DNA of the fresh muscle, ECM and hydrogel derived from muscle was extracted using a DNeasy^® Kit (QIAGEN). Briefly, samples were digested in lysis buffer T1 and proteinase-K at 60°C for 24 h, and DNA was isolated according to kit instructions. NanoDrop (Thermo Scientific) was used to measure eluted DNA. Muscle tissue, ECM and hydrogel were analyzed by SDS–poly-acrylamide gel electrophoresis (SDS–PAGE) to assess protein content according to standard protocol. Gels were stained with Coomassie blue and visualized with an imaging scanner (Licor Odyssey). In addition, western blot was employed to detect TSP2 and collagen type I according to standard protocol. Briefly, equal amounts of protein extracts (50 μg /lane) were separated by SDS-PAGE and transferred to the nitrocellulose membrane (Bio-Rad). Custom TSP-2 antibody (1:250, GenScript) and Collagen I (1:1000, AB765P, EMD Millipore) were employed in conjunction with a secondary antibody conjugated with the fluorescent label (a21109, Invitrogen) diluted 1:1000 in TBST. Finally, the membranes were washed with TBST six times and observed by Infrared Imaging System (Licor Odyssey CLx).

Morphology and Mechanical Properties of the Hydrogels.

SEM was employed to assess the microstructure for the two hydrogel types. Firstly, hydrogels were fixed with 2.5% glutaraldehyde. Specimens were then dehydrated by incubating in an ethanol gradient (i.e., 30, 50, 70, 90 and 100% ethanol). After drying, the specimens were coated with Iridium at a thickness of 8 nm and observed by SEM (SU7000, Hitachi). Fibril curvature and diameter were calculated by Image J analysis of SEM images.

Storage modulus and loss modulus for both hydrogels at 10mg/mL were determined by an AR2000 rheometer (TA Instruments) with a 25 mm parallel plate geometry. Gap height was set to 700 μm. ECM pre-gel was pipetted onto the rheometer plate, which was maintained at a temperature of 10 °C using a Peltier temperature controller. Mineral oil was added to the edge to reduce evaporation of the samples and temperature was increased stepwise by 1°C and allowed to stabilize for 15 s before measurements were taken. This was repeated until the temperature reached 37 °C and maintained to induce gelation. Measurements were done at frequency of 1 Hz and 1% strain.

Proteomics.

Dried ECM was prepared as described above, immersed in PBS and digested with Lys-C and trypsin. Subsequent sample processing and data analysis was performed by the Keck Mass Spectrum and Proteomics Resource Laboratory at Yale University. A volcano plot was generated to show the proteomic differences between WT and TSP2KO decellularized muscle. Two histogram plots were generated based on the abundance of collagen and ECM proteins in the muscle.

Cell Interactions with Hydrogels.

Cells proliferation on the surface of hydrogels was examined to test cytocompatibility. A total of 2×10³ C2C12 muscle cells, NIH3T3 fibroblasts, or HUVECs were seeded and then cultured for 5 days on either WT or TSP2KO derived hydrogels. The proliferation was analyzed every day for 5 days using Cell Counting Kit-8 (CCK-8, ab228554, Abcam). In addition, cells were fixed in 4% paraformaldehyde (PFA, J.T. Baker), and then stained with DAPI (Invitrogen) and rhodamine-phalloidin (Invitrogen).

To examine cell spreading, 1×10⁵ C2C12 cells were seeded and cultured on a 10 mg/mL WT or TSP2KO hydrogel for 2 or 4. Similarly, 1×10⁵ NIH3T3 cells were seeded on a 2 mg/mL WT or TSP2KO hydrogel for 2 h. Hydrogels were rinsed with PBS to remove the unattached cells, and then the specimens were fixed and dehydrated for SEM analysis as described above.

To examine cell invasion, a 10 mg/mL pre-gel was added into the top chamber of a Transwell (Corning) and placed in an incubator at 37 °C for 1h to allow gelation. Subsequently, 5×10⁴ C2C12 cells in 100 μL serum-free media were seeded into the top chamber and culture media with 10% FBS was added to the bottom chamber. Transwells were placed at 37 °C for 6, 8, 12, 48 or 72 h. In addition, 5×10⁴ NIH3T3 cells were seeded on 2 mg/mL hydrogels at 37 °C for 4 and 12 h as described above. At each time point, samples were washed with 1x PBS for 5 min and then fixed with z-fix overnight and then preserved in 70% ethanol. Fixed hydrogels were processed and embedded in paraffin for sectioning. Sections were stained with H&E according to standard protocols and the depth of C2C12 or NIH3T3 cell invasion calculated by Image J.

To evaluate their pro-angiogenic potential, 1×10⁴ HUVECs were seeded on WT or TSP2KO hydrogel (10 mg/mL) for 24 h. Samples were then washed to remove the unattached cells, fixed with 4% PFA overnight, washed three times with PBS and then stained with DAPI (blue) and rhodamine-phalloidin (red) as described above. Numbers and diameters of formed tubes were calculated by Image J from fluorescent images.

Murine immortalized bone marrow macrophages (iBMM) were cultured in IMDM media (Gibco) containing 20% FBS (Gibco), 2.5 mM L-glutamine (Gibco), 1% Penicillin-Streptomycin (CAISSON), and 50 ng/mL M-CSF (R&D Systems). For the polarization assay, 2×10⁵ iBMM were placed in wells (6-well plate) coated with either WT or TSP2KO hydrogel for 3, 7 and 14 days. Cells were visualized by fluorescence following staining with DAPI and rhodamine phalloidin as described above. In addition, qPCR was used to analyze the expression of CD 86, IL-1 β, TNF-α, TGF-β, IL-10, and Arg1. GAPDH was used as an internal control. (Supplementary Table S1).

Foreign Body Response Surgery.

12-week-old C57BL/6J mice were anesthetized with 4% isoflurane and received subcutaneous injections of 250 μL of pre-gel WT or TSP2 KO solution (kept on ice) in the dorsal region. Each mouse received two contralateral injections representing each genotype. Implants were excised en bloc at 5 or 10 days, fixed in Z-fix and embedded in paraffin for sectioning. Sections were stained with H&E, and immunohistochemistry for CD31(1:50, AF3628, R&D Systems), α-SMA (1:200, ab5694, Abcam), Neurofilament H (1:200, AB1989, EMD Millipore), Vimentin (1:500, AB5733, EMD Millipore), F4/80 (1:200, NB600–404SS, Novus) and CD 3e (1:200, 553058, BD Pharma) with the Vector, ABC kit and Peroxidase substrate kit. For analysis of cell penetration, eight 20× images per implant were analyzed.

Volumetric Muscle Loss Surgery.

Twelve-week-old wild-type C57BL/6J mice were anesthetized with 4% isoflurane and maintained under 2.5% isoflurane. Hair was removed from the lower extremity. After ethanol sterilization of the surrounding skin, a 1.5-cm incision was created between the knee and hip joint to access the quadriceps femoris muscle. A 2×7×2 mm piece of the tibialis anterior muscles was removed by surgical scissors. After muscle removal, 200 μl WT or TSP2 KO hydrogel were placed directly into the injury site (five mice per group and time point), and then muscle and skin incisions were sutured layer by layer using 8–0 suture (J975G, VICRYL^™, ETHICON^®) and 6–0 suture (8695G, PROLENE^™, ETHICON^®), respectively. Untreated mice were used as control.

At 3, 7, 14 and 28 post-surgery days animals were euthanized, and the specimens were excised, processed for histological analysis and stained with H&E, Trichrome, and Sirius Red staining. Additionally, samples were analyzed by immunohistochemistry with anti-Laminin (1:200, ab11575, Abcam), anti-Vimentin (1:500, AB5733, EMD Millipore), anti-F4/80 (1:200, NB600–404SS, Novus), anti-CD3e (1:200, 553058, BD Pharma), anti-CD31 (1:50, AF3628, R&D Systems), anti-αSMA (1:50, AF3628, R&D Systems) and anti-Neurofilament H (1:200, AB1989, EMD Millipore) antibodies. Furthermore, immunofluorescent staining with anti-PAX7 (1:500, ab187339, Abcam) and anti-Laminin (1:500, ab11575, Abcam) was performed to visualize active remodeling. ImageJ was used to quantify the number of cells and CD31+ - αSMA+ lumens (20 images per implant, n=5). Neurocytes in the newly formed tissues were confirmed with anti-Neurofilament H antibody (1:1000, AB1989, EMD Millipore) and Western blotting. GAPDH was used as an internal control (1:1000, 14C10, Cell Signaling). For Western blots, the integrated density of protein bands was analyzed by Image J (n=3).

Statistical Analysis.

Data are expressed as mean ± standard error of the mean (SEM). All statistical analyses of data with more than two samples were conducted using one-way ANOVA with Tukey’s multiple comparison test. For analysis of cell penetration into WT, and TSP-2 KO hydrogels at various depths, a two-way ANOVA with Tukey’s multiple comparison test was used. Values of p < 0.05 were considered statistically significant.

Highlights.

• Creation and characterization of muscle-derived extracellular matrix hydrogels from TSP2 KO mice shows altered composition, assembly, and biomechanical properties when compared to WT.

• TSP2 KO hydrogels allow greater myocyte spreading and invasion as well increased tube formation by endothelial cells in vitro.

• In vivo study showed excellent foreign body response of TSP2 KO hydrogels associated with enhanced cell invasion, innervation, and angiogenesis.

• TSP2 KO hydrogels were used to treat VML injury in mice and were found to induce greater recruitment of repair cells and enhanced tissue remodeling.